Injection Of Arachidonic Acid Research Articles

Anti-inflammatory and antinociceptive effects of Aloysia gratissima leaves essential oil: An in vivo study

Background and aimAloysia gratissima is used in popular medicine as an analgesic and sedative. However, studies on its biological activities are still scarce. Therefore, this work aimed to evaluate the antinociceptive and anti-inflammatory effect of A. gratissima leaves essential oil (OAG) in mice. Experimental procedureThe OAG was obtained by hydrodistillation and its composition was determined by gas chromatography-mass spectrometry. The sesquiterpenes pinocamphone, β-pinene, and guaiol were the major constituents of OAG. The effect of OAG (1, 10, and 30 mg/kg, p.o.) was assessed by pre-treating male mice 1h before the formalin assay, carrageenan-induced peritonitis test, or the paw edema induced by arachidonic acid (AA). The acute toxicity of OAG (2000 mg/kg, p.o.) was also investigated. Results and conclusionThe minimum effective dose of OAG in the formalin test was 1 mg/kg. This dose did not affect the locomotor activity of mice. The OAG decreased the number of leukocytes/mm³ in the peritoneal exudate of mice and reduced paw edema 45 min after the arachidonic acid injection. OAG treatment elicited a reduction in NPSH levels in the paw tissue and increased NPSH levels in mice blood plasma and did not cause mice mortality. The OAG is devoid of acute toxicity and shows anti-inflammatory activity, which can be related to the presence of pinocamphone, guaiol, and β-pinene and is mediated, at least in part, by mechanisms that involve the participation of NPSH.

The electrocardiographic changes generated by centrally applied arachidonic acid in rats

Arachidonic acid (AA) and its metabolites have multifunctional regulatory effects on the central nervous system. Our previous reports disclosed that centrally injected AA organized the cardiovascular system in normal or hypotensive conditions by regulating the central and peripheral mechanism. In the light of the knowledge of the potential cardiovascular effects of AA, the current study aimed to investigate the effects of intracerebroventricular (ICV) injected AA on the electrocardiography (ECG) of the anesthetized rats. The adult Sprague Dawley rats were anesthetized with ketamine and xylazine mixture (50 mg/kg and 20 mg/kg; i.m., respectively). Under the anesthesia, the guide cannula was inserted into the left lateral ventricle of the rats. The ECG traces obtained from the lead II were written by placing electrodes on the limbs of the rats. Centrally injected AA (150 μg; ICV) statistically significantly (p<0.05) caused to the lengthening of the ECG waves and intervals, resulting in a decrease in the heart rate of the rats without changing the ECG waveforms, the amplitude, and also the isoelectric line. The obtained results clearly show that centrally injection of AA caused the deceleration in the heart electrical activity. The deceleration in the electrical activity of the heart caused to show bradycardia in the rats by extending the duration of the ECG waves and intervals.

Biosynthetic pathway of arachidonic acid in Spodoptera exigua in response to bacterial challenge

Eicosanoids play crucial roles in mediating insect immune responses. In vertebrates, phospholipase A2 (PLA2) releases arachidonic acid (AA) from phospholipids (PLs) for biosynthesis of various eicosanoids. However, little AA is found in PLs of lepidopteran insects. Spodoptera exigua, a lepidopteran insect, is known to use eicosanoids to mediate immunity. Although AA was not detected in PLs of hemocytes and fat body (two immune tissues) of naïve larvae, it was detected at small but significant level after bacterial infection, suggesting induction of AA biosynthesis for immunity. Based on a mammalian AA biosynthetic pathway, this study hypothesizes that AA is synthesized from C18 polyunsaturated fatty acid (PUFA) precursor by subsequent desaturation and elongation reactions because PLs of S. exigua larvae are rich in linoleic acid. After inhibiting PLA2 activity to prevent release of free fatty acids, different PUFA precursors were injected to S. exigua larvae followed by assessment of eicosanoid-mediated cellular immune response. ω-6 PUFAs were effective in inducing immune response whereas α-linolenic acid (an ω-3 PUFA) was not. Several fatty acyl desaturases (SeDESs) have been predicted from S. exigua transcriptomes. Specific inhibitors against Δ5 or Δ6 DESs inhibited eicosanoid-mediated immune responses. Furthermore, RNA interference (RNAi) specific to Δ5 or Δ6 DES genes significantly suppressed eicosanoid-mediated immune responses. Four very long chain fatty acid elongase genes (SeEloV-A ∼ SeEloV-D) were predicted. Among respective RNAi treatments of these genes, only one RNAi treatment specific to type 5 elongase (SeEloV-B) suppressed eicosanoid-mediated immune response. These results suggest that S. exigua larvae can synthesize AA from linoleic acid via Δ5- and Δ6-desaturations by SeDESs along with chain elongation by SeEloV-B. Finally, this study showed significant fitness cost of uncontrolled AA biosynthesis. AA injection alone without bacterial challenge significantly induced both cellular and humoral immune responses. This unnecessary energy expense due to free AA resulted in reduced pupal size and decreased adult egg production. The detrimental effect of free AA explains physiological significance of little AA content in lepidopteran insects except for life-or-death situation such as pathogen infection.

The inducible blockage of RNAi reveals a role for polyunsaturated fatty acids in the regulation of dsRNA-endocytic capacity in Bactrocera dorsalis

Exogenous double-stranded RNA (dsRNA) can trigger gene silencing through the RNA interference (RNAi) pathway. Our previous research established that Bactrocera dorsalis can block RNAi after an initial priming of exposure to dsRNA. However, the mechanism underlying this phenomenon is not yet fully understood. Here, we demonstrate that fatty acid biosynthesis and metabolism pathways play important roles in the blockage of RNAi induced by dsRNA priming. The ratio of linoleic acid (LA) to arachidonic acid (AA) was significantly increased in the hemolymph of B. dorsalis following dsRNA priming, and further, the endocytosis of dsRNA into the midgut cells of B. dorsalis was inhibited in these samples. The expression levels of most genes involved in the fatty acid biosynthesis and metabolism pathways were altered following priming with dsRNA. Furthermore, altering the composition of fatty acids via the injection of AA can facilitate the uptake of ingested dsRNA into the midgut cells of Drosophila melanogaster and successfully induce an RNAi effect, which cannot be achieved via feeding in fruit flies. Our results suggest that polyunsaturated fatty acids are involved in the regulation of the dsRNA-endocytic ability in B. dorsalis.

Role of prostaglandins in spinal transmission of the exercise pressor reflex in decerebrated rats

Previous studies found that prostaglandins in skeletal muscle play a role in evoking the exercise pressor reflex; however the role played by prostaglandins in the spinal transmission of the reflex is not known. We determined, therefore, whether or not spinal blockade of cyclooxygenase (COX) activity and/or spinal blockade of endoperoxide (EP) 2 or 4 receptors attenuated the exercise pressor reflex in decerebrated rats. We first established that intrathecal doses of a non-specific COX inhibitor Ketorolac (100μg in 10μl), a COX-2-specific inhibitor Celecoxib (100μg in 10μl), an EP2 antagonist PF-04418948 (10μg in 10μl), and an EP4 antagonist L-161,982 (4μg in 10μl) effectively attenuated the pressor responses to intrathecal injections of arachidonic acid (100μg in 10μl), EP2 agonist Butaprost (4ng in 10μl), and EP4 agonist TCS 2510 (6.25μg in 2.5μl), respectively. Once effective doses were established, we statically contracted the hind limb before and after intrathecal injections of Ketorolac, Celecoxib, the EP2 antagonist and the EP4 antagonist. We found that Ketorolac significantly attenuated the pressor response to static contraction (before Ketorolac: 23±5mmHg, after Ketorolac 14±5mmHg; p<0.05) whereas Celecoxib had no effect. We also found that 8μg of L-161,982, but not 4μg of L-161,982, significantly attenuated the pressor response to static contraction (before L-161,982: 21±4mmHg, after L-161,982 12±3mmHg; p<0.05), whereas PF-04418948 (10μg) had no effect. We conclude that spinal COX-1, but not COX-2, plays a role in evoking the exercise pressor reflex, and that the spinal prostaglandins produced by this enzyme are most likely activating spinal EP4 receptors, but not EP2 receptors.

Activation of the central histaminergic system mediates arachidonic-acid-induced cardiovascular effects.

The aim of this study was to explain the involvement of the central histaminergic system in arachidonic acid (AA)-induced cardiovascular effects in normotensive rats using hemodynamic, immunohistochemistry, and microdialysis studies. Intracerebroventricularly (i.c.v.) administered AA (0.25, 0.5, and 1.0 μmol) induced dose- and time-dependent increases in mean arterial pressure and decreased heart rate in conscious normotensive Sprague-Dawley rats. Central injection of AA (0.5 μmol) also increased posterior hypothalamic extracellular histamine levels and produced strong COX-1 but not COX-2 immunoreactivity in the posterior hypothalamus of rats. Moreover, the cardiovascular effects and COX-1 immunoreactivity in the posterior hypothalamus induced by AA (0.5 μmol; i.c.v.) were almost completely blocked by the H2 receptor antagonist ranitidine (50 and 100 nmol; i.c.v.) and partially blocked by the H1 receptor blocker chlorpheniramine (100 nmol; i.c.v.) and the H3-H4 receptor antagonist thioperamide (50 and 100 nmol; i.c.v.). In conclusion, these results indicate that centrally administered AA induces pressor and bradycardic responses in conscious rats. Moreover, we suggest that AA may activate histaminergic neurons and increase extracellular histamine levels, particularly in the posterior hypothalamus. Acting as a neurotransmitter, histamine is potentially involved in AA-induced cardiovascular effects under normotensive conditions.

English

This study aimed&nbsp;to investigate the effect of polydatin against thrombosis and its possible mechanisms. The methods of injection of arachidonic acid into mouse tail vein electrically stimulated carotid thrombosis in rats, and the rats&rsquo; inferior vena ligation were used to evaluate the antithrombotic effects of polydatin. Platelet aggregation was tested by use of Born&rsquo;s method, and platelet cytosolic calcium was determined by use of Fura-2/AM. Thromboxane B2&nbsp;and 6-keto-prostaglandin F1&alpha;&nbsp;level was monitored by the immuno-assay, while Rosette assay and Born&rsquo;s method were used to observe platelet-neutrophil interactions. The results show that polydatin had evident antithrombotic effects in the multiple-thrombosis models; the mechanisms may be closely related to its anti-platelet aggregation, which results in decrease of platelet cytosolic calcium and plasma thromboxane B2, while increasing plasma 6-keto-prostaglandin F1&alpha;&nbsp;Level and suppressing of platelet-neutrophil interactions. &nbsp; Key words:&nbsp;Polydatin, thromboxane A2, prostacyclin, platelet, neutrophil, cytosolic calcium.

Effects of Copper-aspirin Complex on Platelet Aggregation and Thrombosis in Rabbits and Mice

The effects of intragastric and intraduodenal copper-aspirin complex on rabbit platelet aggregation were observed by Born's method. Myers's method was used to evaluate the antithrombotic effect of copper-aspirin complex in mice. In-vitro copper-aspirin complex selectively inhibited arachidonic acid-induced platelet aggregation with an IC50 value (concentration resulting in 50% inhibition) of 13.2 microM (95% confidence limits 9.1-16.8 microM). Copper-aspirin complex (10 mg kg(-1) given intragastrically or intraduodenally) was more potent than aspirin in inhibiting arachidonic acid-induced platelet aggregation. Copper-aspirin complex (10 mg kg(-1)) had a stronger inhibitory effect and a longer duration of action when given intragastrically than when given intraduodenally. It was shown by radioimmunoassay that copper-aspirin complex significantly reduced the level of thromboxane B2 in plasma while markedly increasing that of 6-ketoprostaglandin F1alpha (6keto-PGF1alpha). Copper-aspirin complex (10 mg kg(-1) given intragastrically for 7 days) significantly reduced mouse mortality caused by intravenous injection of arachidonic acid. The results suggest that both in-vitro and in-vivo copper-aspirin complex is more potent in selectively inhibiting arachidonic acid-induced platelet aggregation than aspirin. When given intragastrically the complex has a more potent antiplatelet effect and a longer duration of action than when given intraduodenally. The antithrombotic effect of the complex was more potent than that of aspirin.

Central mechanism underlying pressor and bradycardic effect of intracerebroventricularly injected arachidonic acid

The aim of the current study was to determine the central cyclooxygenase (COX) pathway and central thromboxane signaling in the cardiovascular effects evoked by arachidonic acid (AA). As a main control for the study, different doses of AA (75, 150, or 300µg) were administered intracerebroventricularly (i.c.v.). Centrally injected AA dose- and time-dependently increased mean arterial pressure and decreased heart rate in conscious normotensive Sprague-Dawley rats. The maximal cardiovascular effects of AA were observed at min 10 of the injection and lasted almost 30min. To investigate the central mechanism of the AA-induced cardiovascular effect in conscious normotensive animals, pretreatment with nonselective COX inhibitor indomethacin (200µg; i.c.v.), thromboxane A2 (TXA2) synthesis inhibitor furegrelate (250 or 500µg; i.c.v.), or TXA2 receptor antagonist SQ-29548 (8 or 16µg; i.c.v.) was carried out 15min before AA (150µg; i.c.v.) injection. While indomethacin completely prevented the pressor and bradycardic responses to AA, furegrelate and SQ-29548 attenuated these effects in part in awake normotensive rats. In conclusion, these findings suggest that the pressor and bradycardic cardiovascular effects of centrally injected AA are dependent on COX activity being totally central and the TXA2 signaling pathway being subsequently central, at least in part.

CARDIOVASCULAR EFFECTS OF CENTRALLY ADMINISTERED ARACHIDONIC ACID IN HAEMORRHAGE‐INDUCED HYPOTENSIVE RATS: INVESTIGATION OF A PERIPHERAL MECHANISM

1. The aims of the present study were to determine the cardiovascular effects of arachidonic acid (AA) and to investigate the peripheral mechanisms mediating these effects in haemorrhage-induced hypotensive rats. 2. Acute haemorrhage was induced by withdrawing a total volume of 2.2 mL blood/100 g bodyweight over a period of 10 min. Rats were then injected with 75-300 microg, i.c.v., AA and cardiovascular changes were monitored over the next 60 min. Plasma catecholamine and vasopressin levels, as well as plasma renin activity (PRA), were measured 10 min after injection of 150 microg AA in haemorrhage-induced hypotensive awake rats. In addition, rats were pretreated with saline (1 mL/kg, i.v.), the vasopressin V(1) receptor antagonist [beta-mercapto-beta,beta-cyclopentamethylenepropionyl(1),O-Me-Tyr(2),Arg(8)]-vasopressin (10 microg/kg, i.v.), the alpha(1)-adrenoceptor antagonist prazosin (500 microg/kg, i.v.), the non-specific angiotensin II receptor antagonist saralasin (250 microg/kg, i.v.) or a combination of these three antagonists 5 min before injection of AA (150 microg, i.c.v.). The effects of these antagonists on responses to AA were determined. 3. Arachidonic acid caused dose- and time-dependent increases in mean arterial pressure and heart rate and reversed hypotension in haemorrhaged rats. Haemorrhage itself produced an increase in plasma catecholamine and vasopressin levels, as well as PRA; injection of AA produced further increases in these parameters, ranging from 39-123%, under hypotensive conditions. Under hypotensive conditions, pretreatment of rats with all three receptor antagonists produced similar partial blockade of the pressor response to AA, but not the increase in heart rate. Moreover, combined administration of all three receptor antagonists prior to the i.c.v. injection of 150 microg AA completely abolished the pressor response to AA in haemorrhage-induced hypotensive rats. 4. These results indicate that centrally administered AA reverses hypotension by increasing blood pressure and heart rate in the hypotensive setting. The observed increases in plasma catecholamine and vasopressin levels, as well as PRA, mediate the pressor response to AA in haemorrhage-induced hypotensive rats.

ASIC3, a sensor of acidic and primary inflammatory pain

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular acidosis that are expressed in both central and peripheral nervous systems. Although peripheral ASICs seem to be natural sensors of acidic pain (e.g., in inflammation, ischaemia, lesions or tumours), a direct demonstration is still lacking. We show that approximately 60% of rat cutaneous sensory neurons express ASIC3-like currents. Native as well as recombinant ASIC3 respond synergistically to three different inflammatory signals that are slight acidifications (approximately pH 7.0), hypertonicity and arachidonic acid (AA). Moderate pH, alone or in combination with hypertonicity and AA, increases nociceptors excitability and produces pain suppressed by the toxin APETx2, a specific blocker of ASIC3. Both APETx2 and the in vivo knockdown of ASIC3 with a specific siRNA also have potent analgesic effects against primary inflammation-induced hyperalgesia in rat. Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Intracerebroventricular injection of arachidonic acid and other unsaturated fatty acids induces cyclooxygenase-2 in brain endothelial cells and evokes fever in rats

Intracerebroventricular injection of arachidonic acid and other unsaturated fatty acids induces cyclooxygenase-2 in brain endothelial cells and evokes fever in rats

Arachidonic acid as a retrograde signal controlling growth and dynamics of retinotectal arbors

In the developing visual system, correlated presynaptic activity between neighboring retinal ganglion cells (RGC) stabilizes retinotopic synapses via a postsynaptic NMDAR (N-methyl-D-aspartate receptor)-dependent mechanism. Blocking NMDARs makes individual axonal arbors larger, which underlies an unsharpened map, and also increases branch turnover, as if a stabilizing factor from the postsynaptic partner is no longer released. Arachidonic acid (AA), a candidate retrograde stabilizing factor, is released by cytoplasmic phospholipase A2 (cPLA2) after Ca(2+) entry through activated NMDARs, and can activate presynaptic protein kinase C to phosphorylate various substrates such as GAP43 to regulate cytoskeletal dynamics. To test the role of cPLA2 in the retinotectal system of developing zebrafish, we first used PED6, a fluorescent reporter of cPLA2 activity, to show that 1-3 min of strobe flashes activated tectal cPLA2 by an NMDAR-dependent mechanism. Second, we imaged the dynamic growth of retinal arbors during both local inhibition of tectal cPLA2 by a pharmacological inhibitor, arachidonic tri-fluoromethylketone, and its suppression by antisense oligonucleotides (both injected intraventricularly). Both methods produced larger arbors and faster branch dynamics as occurs with blocking NMDARs. In contrast, intraocular suppression of retinal cPLA2 with large doses of antisense oligos produced none of the effects of tectal cPLA2 inhibition. Finally, if AA is the retrograde messenger, the application of exogenous AA to the tectum should reverse the increased branch turnover caused by blocking either NMDARs or cPLA2. In both cases, intraventricular injection of AA stabilized the overall branch dynamics, bringing rates down below the normal values. The results suggest that AA generated postsynaptically by cPLA2 downstream of Ca(2+) entry through NMDARs acts as a retrograde signal to regulate the dynamic growth of retinal arbors.

Effect of hemoglobin vesicle, a cellular-type artificial oxygen carrier, on middle cerebral artery occlusion- and arachidonic acid-induced stroke models in rats

Hemoglobin vesicle (HbV), which is also called liposome-encapsulated hemoglobin, functions as a hemoglobin-based oxygen carrier and is expected to be utilized in emergency situations including hemorrhagic shock and several kinds of ischemic diseases. In the present study, we evaluated the efficacy of HbV for improving stroke-related symptoms induced by middle cerebral artery (MCA) occlusion/reperfusion and an intra-internal carotid arterial injection of arachidonic acid (AA) in rats. When HbV (10 mL/kg, i.v.) was administered to rats immediately after the MCA occlusion, it reduced the cerebral infarct volumes of the cortex and total of the cortex plus sub-cortex significantly as compared with saline as a vehicle. In AA-induced stroke model, HbV (10 mL/kg, i.v.) improved the motor dysfunction score and inhibited the increase in cerebral water content suggesting it could suppress cerebral edema. These results strongly suggest that HbV would provide a novel beneficial option for the treatment of stroke, especially acute ischemic stroke.

Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella

Abstract Teratocytes are cells that originate from the extra‐embryonic tissues of some hymenopteran parasitoids, typically dissociate upon hatching, and develop in the host haemolymph. They are considered to be involved in parasitoid larval nutrient uptake, host immunosuppression and/or repression of competing parasitoid development. Teratocytes of the parasitoid, Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae) are found in its natural host, Plutella xylostella (Linnaeus) (Lepidoptera: Yponomeutidae) and can be cultured in vitro. The present study demonstrates that teratocytes of C. plutellae possess a significantly depressive effect on host cellular immunity. When the hosts are preinjected with 200 cultured teratocytes (corresponding to the normal number of teratocytes released during wasp hatching), haemocyte nodulation is inhibited by approximately 40%, with younger teratocytes being more potent than older ones. Similarly, the medium in which teratocytes are cultured has similar immunosuppressive properties. In comparison, calyx fluid extracted from the C. plutellae ovary also has an immunosuppressive effect on P. xylostella. These two maternal (calyx fluid) and embryonic (teratocytes) factors are additive and result in a reduced level of nodule formation equivalent to that induced by natural parasitization. However, the immunosuppression of the parasitized P. xylostella does not appear to be due to inhibition of phospholipase A2, an immune mediator, because injection of arachidonic acid failed to restore haemocyte nodulation capability.

Effects of intracerebroventricular injections of free fatty acids, lysophospholipids, or platelet activating factor in a mouse model of orofacial pain

The present study was carried out to determine the effects of central nervous free fatty acids, lysophospholipids, or platelet activating factor (PAF), in a mouse facial carrageenan injection model of orofacial pain. Mice that received intracerebroventricular (I.C.V.) injection of arachidonic acid or oleic acid showed significantly reduced allodynia and behavioral responses to von Frey hair stimulation of a carrageenan-injected area of the face, at 8 h post-injection, compared to controls that received I.C.V. injection of vehicle. In contrast to free fatty acids, increased responses were observed in mice at 72 h after I.C.V. lysophosphatidic acid or lysophosphatidylcholine injection, and at 8 and 24 h after PAF injection, compared vehicle injected controls. Information regarding pro-nociceptive effect of specific brain lipids may be a useful basis for further studies to explore mechanism.

Intracerebroventricular injection of phospholipases A 2 inhibitors modulates allodynia after facial carrageenan injection in mice

The present study was carried out, using inhibitors to secretory phospholipase A 2 (sPLA 2, 12-epi-scalaradial), cytosolic phospholipase A 2 (cPLA 2, AACOCF 3), or calcium-independent phospholipase A 2 (iPLA 2, bromoenol lactone), to compare possible contributions of central nervous PLA 2 isoforms to the development of allodynia after facial carrageenan injection in mice. C57BL/6J (B6) mice showed increased responses to facial stimulation using a von Frey hair (1 g force), at 8 h, 1 day, and 3 days after facial carrageenan injection. On the other hand, BALB/c mice did not show increased responses at any of the time points. In both B6 and BALB/c mice, intracerebroventricular injection of inhibitors to each of the three PLA 2 isoforms significantly reduced responses to von Frey hair stimulation at 8 h and 1 day after facial carrageenan injection, but at 3 days after injection, only the sPLA 2 inhibitor had an effect. Since BALB/c mice did not show increased responses after facial carrageenan injection, the reduction in responses actually indicates that there is loss of normal sensitivity to von Frey hair stimulation after intracerebroventricular injection of each of these inhibitors, in this strain of mice. The effects of PLA 2 inhibitors are unlikely to be due simply to inhibition of arachidonic acid generation, since intracerebroventricular injection of arachidonic acid also had an anti-nociceptive effect. The above results support an important role of central nervous PLA 2s in neurotransmission and pain transmission.

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua

An entomopathogenic bacterium, Xenorhabdus nematophila, is known to depress hemocyte nodule formation of target insects by inhibiting eicosanoid biosynthesis. This study analyzed the inhibitory effect of X. nematophila on the humoral immunity of the target insects and tested its association with the host eicosanoid pathway. Plasma collected from the fifth instar larvae of Spodoptera exigua, when they were injected with X. nematophila, did not show antibacterial activity against Escherichia coli by a growth inhibition zone assay. In comparison, heat-killed X. nematophila induced significant antibacterial activity in the plasma. The antibacterial humoral activity was further demonstrated by examining a specific potent antibacterial peptide, cecropin. Two cecropin genes (‘A’ and ‘B’) were partially cloned from the fifth instar larvae of S. exigua by conserved degenerate primers using nested reverse transcriptase-polymerase chain reaction (RT-PCR). They showed high homologies with known cecropins from other lepidopteran species. Northern analysis using the cecropin probe showed that the injection of the heat-killed X. nematophila induced significant expression of a cecropin mRNA transcript (≈1.1 kb), but the larvae injected with the live bacteria did not show the corresponding transcript. Injection of arachidonic acid did not rescue the inhibition of X. nematophila based on either antibacterial activity or cecropin gene expression. The addition of dexamethasone, a specific phospholipase A 2 inhibitor, did not inhibit antibacterial activity or cecropin gene expression when the larvae were injected with heat-killed X. nematophila. These results suggest that X. nematophila inhibits the antibacterial humoral immune reaction as well as the cellular immune reaction in S. exigua and that the inhibition of X. nematophila on the expression of the antibacterial peptide is not associated with inhibition of the eicosanoid pathway.

Role of cyclooxygenase-2 in the generation of vasoactive prostanoids in the rat pulmonary and systemic vascular beds.

Prostanoid synthesis by the cyclooxygenase (COX)-2 pathway plays an important role in inflammation, and recent studies have shown the presence of COX-2 in the normal rat lung. However, the role of COX-2 in the generation of vasoactive prostanoids in the rat is uncertain. In the present study, the hypothesis that synthesis of vasoactive prostanoids via the COX-2 pathway can alter pulmonary and systemic vascular resistance was investigated, and the effects of selective COX-2 inhibitors on pulmonary and systemic responses to the prostanoid precursor arachidonic acid were examined in the anesthetized rat with a recently developed right-heart catheterization technique. Injections of arachidonic acid caused dose-related increases in pulmonary vascular resistance and decreases in systemic vascular resistance. These responses were attenuated by selective COX-2 inhibitors and a selective COX-1 inhibitor, whereas responses to exogenous prostanoids were not altered. Nimesulide or NS-398 did not alter arachidonic acid-induced platelet aggregation in rat platelet-rich plasma. Western blot analysis and immunostaining showed the expression of both COX isoforms in the rat lung. The results of these experiments suggest that arachidonic acid is converted into vasoactive prostanoids by the COX-2 and COX-1 pathway in the pulmonary and peripheral vascular beds in the rat and that TXA2 is a major prostanoid formed in the normal rat lung.

Origins of Prostaglandin E2: Involvements of Cyclooxygenase (COX)-1 and COX-2 in Human and Rat Systems

Prostaglandin (PG) E2 is a major cyclooxygenase (COX) product at inflammatory sites where it contributes to local increases in blood flow, edema formation, and pain sensitization. Using rats in vivo and rat and human blood in vitro, we have examined the roles of COX-1 and COX-2 in the production of PGE2. In anesthetized rats treated with bacterial lipopolysaccharide (LPS) to induce the expression of COX-2, the marked increase in PGE2 production that followed bolus intravenous injection of arachidonic acid (3 mg x kg(-1)) was strongly inhibited by diclofenac but largely unaffected by the COX-2-selective inhibitor DFP (5,5- dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone). In rat blood in vitro, aspirin strongly inhibited the production of PGE2 that followed either acute exposure to calcium ionophore, A23187 (calcimycin) (50 microM, 15 min), or incubation with LPS for 18 h. In contrast, human whole blood only produced significant levels of PGE2 when incubated with LPS. Rat leukocytes expressed COX-2 and produced PGE2 when exposed to LPS but not when acutely stimulated with A23187. Rat platelets, but not human platelets, also produced significant amounts of PGE2 when acutely stimulated with A23187. These data show that when exposed to an inflammatory stimulus, rat whole blood produces increased levels of PGE2 through induction of COX-2 in blood leukocytes. Rat blood, unlike human blood, may also produce copious amounts of PGE2 via the actions of COX-1 enzyme constitutively present in platelets. These data may well explain why in rats COX-2-selective inhibitors have been reported not to produce the full anti-inflammatory effects associated with standard nonsteroid anti-inflammatory drugs.